Stroke is the third leading cause of death in the United States and the main cause of long-term disability. The risk of stroke increases significantly with age, and the average age of Veterans will continue to increase as Veterans from the Vietnam era continue to age. The overall goals of this project are to develop and characterize novel therapeutic strategies that will improve outcome and reduce long-term disability after stroke. Cerebral ischemia and subsequent reperfusion initiates a cascade of events that can lead to inflammation and secondary neuronal damage resulting in an increased extent of infarct and poorer clinical outcome. The complement system plays a central role in the pathophysiology of ischemic stroke. We propose to investigate the pathogenic, as well as the protective mechanisms of complement that are involved in cerebral injury after stroke, and to develop and characterize novel complement inhibitory strategies for reducing secondary tissue injury and improving cognitive and long-term functional recovery. The proposed studies are focused on the characterization of targeted complement inhibitors in pre-clinical murine models of stroke. We have demonstrated the significant benefits of targeted versus systemic complement inhibition in models of CNS injury, including ischemic stroke, and here we propose to characterize second-generation targeted complement inhibitors that target to injured brain tissue after ischemia. Our targeting strategy consists of linking different types of complement inhibitor to single chain antibody (scFv) targeting moieties. We will characterize different targeting specificities and different types of complement inhibitor in two different models of stroke, and determine short-term and long-term therapeutic outcome in terms of tissue injury, tissue repair and regeneration, and cognitive and functional recovery. In aim 1, we will characterize the recombinant proteins in a mouse model of middle cerebral artery occlusion (MCAO). This is a well characterized model and will allow comparisons with previous and archived data. We will investigate the post-reperfusion treatment window, and the temporal expression of different neoepitopes on the ischemic brain (which may indicate different targeting strategies, depending on when inhibitor is administered after stroke). We will additionally explore stress/injury neoepitopes that are more selective for brain endothelial cells in an attempt to identify additional targets for delivery of therapeutics. In aim 2, we will use the same MCAO model to investigate the effect of the different recombinant proteins in long-term outcome, in terms of tissue injury and repair, cognitive function and functional recovery. These studies will also address the dual role of complement in injury and repair/neuroregeneration. In aim 3, we will characterize the most promising candidate inhibitors in a model of microembolic stroke. The microembolic model allows more control over clot burden, perfusion impairment and neurological deficit. A focus here will be cognitive and functional evaluation and long-term recovery. The model also allows evaluation of spontaneous and therapeutic dissolution of clots in combination with complement inhibitory therapy. This will provide important pre-clinical data since the only currently approved treatment for ischemic stroke is tissue plasminogen activator (tPA).